Intracellular delivery of an antisense oligonucleotide via endocytosis of a G protein-coupled receptor

Gastrin-releasing peptide receptor (GRPR), a member of the G protein-coupled receptor superfamily, has been utilized for receptor-mediated targeting of imaging and therapeutic agents; here we extend its use to oligonucleotide delivery. A splice-shifting antisense oligonucleotide was conjugated to a bombesin (BBN) peptide, and its intracellular delivery was tested in GRPR expressing PC3 cells stably transfected with a luciferase gene interrupted by an abnormally spliced intron. The BBN-conjugate produced significantly higher luciferase expression compared to unmodified oligonucleotide, and this increase was reversed by excess BBN peptide. Kinetic studies revealed a combination of saturable, receptor-mediated endocytosis and non-saturable pinocytosis for uptake of the conjugate. The Km value for saturable uptake was similar to the EC50 value for the pharmacological response, indicating that receptor-mediated endocytosis was a primary contributor to the response. Use of pharmacological and molecular inhibitors of endocytosis showed that the conjugate utilized a clathrin-, actin- and dynamin-dependent pathway to enter PC3 cells. The BBN-conjugate partially localized in endomembrane vesicles that were associated with Rab7 or Rab9, demonstrating that it was transported to late endosomes and the trans-golgi network. These observations suggest that the BBN-oligonucleotide conjugate enters cells via a process of GRPR mediated endocytosis followed by trafficking to deep endomembrane compartments

Evaluating the Specificity of Antisense Oligonucleotide Conjugates: A DNA ARRAY ANALYSIS

Antisense oligonucleotides are potentially powerful tools for selective control of cellular and viral gene expression. Crucial to successful application of this approach is the specificity of the oligonucleotide for the chosen RNA target. Here we apply DNA array technology to examine the specificity of antisense oligonucleotide treatments. The molecules used in these studies consisted of phosphorothioate oligomers linked to the Antennapedia (Ant) delivery peptide. The antisense oligonucleotide component was complementary to a site flanking the AUG of the MDR1 message, which codes for P-glycoprotein, a membrane ATPase associated with multidrug resistance in tumor cells. Using a DNA array of 2059 genes, we analyzed cellular responses to molecules comprised of Ant peptide-oligonucleotide conjugates, as well as to the Ant peptide alone. Besides the expected reduction in MDR1 message level, 37 other genes (approximately 2% of those tested) showed changes of comparable magnitude. The validity of the array results was confirmed for selected genes using Northern blots to assess messenger RNA levels. These results suggest that studies using antisense oligonucleotide technology to modulate gene expression need to be interpreted with caution

A Molecular Umbrella Approach to the Intracellular Delivery of siRNA

A series of diwalled and tetrawalled molecular umbrellas have been synthesized using cholic acid, spermidine, and lysine as starting material. Coupling of these molecular umbrellas to an octaarginine peptide afforded agents that were capable of promoting the transport of small interfering RNA (siRNA) to HeLa cells, as judged by the knockdown of enhanced green fluorescent protein (eGFP) expression. The efficiency of this knockdown was found to increase with an increasing number of facially amphiphilic walls present, and also when a cleavable disulfide linker was replaced with a non-cleavable, maleimido moiety. The knockdown efficiency that was observed for one tetrawalled molecular umbrella-octaargine conjugate was comparable to that observed with a commercially available transfection agent, Lipofectamine 2000, but showed less cytotoxicity

Intracellular delivery of an antisense oligonucleotide via endocytosis of a G protein-coupled receptor

Gastrin-releasing peptide receptor (GRPR), a member of the G protein-coupled receptor superfamily, has been utilized for receptor-mediated targeting of imaging and therapeutic agents; here we extend its use to oligonucleotide delivery. A splice-shifting antisense oligonucleotide was conjugated to a bombesin (BBN) peptide, and its intracellular delivery was tested in GRPR expressing PC3 cells stably transfected with a luciferase gene interrupted by an abnormally spliced intron. The BBN-conjugate produced significantly higher luciferase expression compared to unmodified oligonucleotide, and this increase was reversed by excess BBN peptide. Kinetic studies revealed a combination of saturable, receptor-mediated endocytosis and non-saturable pinocytosis for uptake of the conjugate. The Km value for saturable uptake was similar to the EC50 value for the pharmacological response, indicating that receptor-mediated endocytosis was a primary contributor to the response. Use of pharmacological and molecular inhibitors of endocytosis showed that the conjugate utilized a clathrin-, actin- and dynamin-dependent pathway to enter PC3 cells. The BBN-conjugate partially localized in endomembrane vesicles that were associated with Rab7 or Rab9, demonstrating that it was transported to late endosomes and the trans-golgi network. These observations suggest that the BBN-oligonucleotide conjugate enters cells via a process of GRPR mediated endocytosis followed by trafficking to deep endomembrane compartments

The Biological Effect of an Antisense Oligonucleotide Depends on Its Route of Endocytosis and Trafficking

We demonstrate that the biological effect of an oligonucleotide is influenced by its route of cellular uptake. Utilizing a splice-switching antisense oligonucleotide (SSO) and a sensitive reporter assay involving correction of RNA splicing, we examined induction of luciferase in cells treated either with various concentrations of an unconjugated (“free”) SSO or an SSO conjugated to a bivalent RGD ligand that promotes binding to the αvβ3 integrin (RGD-SSO). Under conditions of equal accumulation in cells, the RGD-SSO consistently had a greater effect on luciferase induction than the unconjugated SSO. We determined that the RGD-SSO and the unconjugated SSO were internalized by distinct endocytotic pathways, suggesting that the route of internalization affects the magnitude of the biological response

Inhibition of MDR1 expression with altritol-modified siRNAs

Altritol-modified nucleic acids (ANAs) support RNA-like A-form structures when included in oligonucleotide duplexes. Thus altritol residues seem suitable as candidates for the chemical modification of siRNAs. Here we report that ANA-modified siRNAs targeting the MDR1 gene can exhibit improved efficacy as compared to unmodified controls. This was particularly true of ANA modifications at or near the 3′ end of the sense or antisense strands, while modification at the 5′ end of the antisense strand resulted in complete loss of activity. Multiple ANA modifications within the sense strand were also well tolerated. Duplexes with ANA modifications at appropriate positions in both strands were generally more effective than duplexes with one modified and one unmodified strand. Initial evidence suggests that the loss of activity associated with ANA modification of the 5′-antisense strand may be due to reduced phosphorylation at this site by cellular kinases. Treatment of drug resistant cells with MDR1-targeted siRNAs resulted in reduction of P-glycoprotein (Pgp) expression, parallel reduction in MDR1 message levels, increased accumulation of the Pgp substrate rhodamine 123, and reduced resistance to anti-tumor drugs. Interestingly, the duration of action of some of the ANA-modified siRNAs was substantially greater than that of unmodified controls. These observations suggest that altritol modifications may be helpful in developing siRNAs with enhanced pharmacological effectiveness

Challenges and opportunities for quantifying roots and rhizosphere interactions through imaging and image analysis

The morphology of roots and root systems influences the efficiency by which plants acquire nutrients and water, anchor themselves and provide stability to the surrounding soil. Plant genotype and the biotic and abiotic environment significantly influence root morphology, growth and ultimately crop yield. The challenge for researchers interested in phenotyping root systems is, therefore, not just to measure roots and link their phenotype to the plant genotype, but also to understand how the growth of roots is influenced by their environment. This review discusses progress in quantifying root system parameters (e.g. in terms of size, shape and dynamics) using imaging and image analysis technologies and also discusses their potential for providing a better understanding of root:soil interactions. Significant progress has been made in image acquisition techniques, however trade-offs exist between sample throughput, sample size, image resolution and information gained. All of these factors impact on downstream image analysis processes. While there have been significant advances in computation power, limitations still exist in statistical processes involved in image analysis. Utilizing and combining different imaging systems, integrating measurements and image analysis where possible, and amalgamating data will allow researchers to gain a better understanding of root:soil interactions

2′-O-HYDROXYALKOXYMETHYLRIBIBONUCLEOSIDE AND THEIR INCORPORATION INTO OLIGORIBONUCLEOTIDES

A simple and efficient method for the preparation of pyrimidine 2′-O-hydroxyethoxymethylribonucleosides and 2′-O-hydmxypropoxymethylribonucleosides has been developed. These modified nucleosides were incorporated into oligoribonucleotides, which were shown to form stable RNA/RNA duplexes. The effect of 2′-O-modification in the antisense and sense strands of small interference RNA was evaluated in multi-drug resistant NIH 3T3 cells

A case of familial isolated hemihyperplasia

BACKGROUND: Hemihyperplasia (hemihypertrophy) is defined as asymmetric body overgrowth of one or more body parts. Hemihyperplasia can be isolated or be part of well-defined syndromes such as in the case of Beckwith-Wiedemann syndrome (BWS). Isolated hemihyperplasia is usually sporadic, but a number of familial occurrences have been described. CASE PRESENTATION: We describe a Tunisian family in which three maternal cousins and their maternal grandfather present with isolated hemihyperplasia. CONCLUSIONS: The etiology of isolated hemihyperplasia is unknown although in BWS, genomic imprinting has been shown to play a role in the asymmetric overgrowth. Given the similarity between these two conditions, it is possible that both may share a common pathogenesis. We also discuss the possible genetic mechanisms leading to the production of hemihyperplasia in this family